Validation of the Glaucoma Filtration Surgical Mouse Model

INTRODUCTIONThe most common reason for failure in

glaucoma filtration surgery (GFS) is scar-ring and fibrosis. Subconjunctival scar-ring at the level of the subconjunctival fi-broblasts often leads to poorly filteringblebs and a subsequent rise in intraocu-lar pressure (IOP) (1). To prevent scar-ring, pharmacological approaches, suchas the use of antifibrotic agents, havebeen attempted in experimental and clin-ical studies. Drugs with proven efficacyin humans include topical corticos-teroids, and intraoperatively applied an-tiproliferatives such as 5-fluorouracil

(5FU) and mitomycin C (MMC) (2–5).MMC, an antibiotic secreted by Strepto-myces caespitosus, acts as an alkylatingagent that crosslinks DNA, thereby in-hibiting DNA synthesis. MMC also in-hibits RNA and protein synthesis and itinteracts with molecular oxygen, whichin turn generates free radical damage toDNA and protein (6). These nonspecificeffects are, however, associated with se-vere and often blinding complicationsthat include hypotonous maculopathy(7), thin, cystic and leaky blebs that areprone to infections, and bleb-related en-dophthalmitis (8,9). Furthermore, GFS

fails in a substantial number of high-riskeyes despite the use of these antiprolifer-ative agents. As a result, the subject andunderstanding of the subconjunctivalwound healing response is far from com-plete, and the quest to find a safer alter-native and more specific antiscarringagent remains a top priority.

To address this area of research, sev-eral animal models, including rat (10,11),rabbit (12–15) and monkey (16,17), havebeen developed to study the clinical andhistological scarring response after GFS.The rabbit has, by far, been the mostpopular animal used for such studiesowing to both the relatively large ocularstructures, allowing ease of surgical ma-nipulation, and cost-effectiveness. How-ever, many aspects of GFS cannot be ex-amined in detail in the rabbit owing tothe limited availability of reagents suchas antibodies, gene expression arrays,and so on. To date, a successful mousemodel for GFS has not been described,

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Validation of the Glaucoma Filtration Surgical Mouse Modelfor Antifibrotic Drug Evaluation

Li-Fong Seet,1 Wing Sum Lee,1 Roseline Su,1 Sharon N Finger,1 Jonathan G Crowston,2 and Tina T Wong1,3,4,5

1Ocular Wound Healing and Therapeutics, Singapore Eye Research Institute, Singapore; 2Centre for Eye Research Australia, RoyalVictorian Eye and Ear Hospital, Melbourne, Australia; 3Glaucoma Service, Singapore National Eye Center, Singapore; 4Department ofOphthalmology, Yong Loo Lin School of Medicine, National University of Singapore; and 5School of Materials Science andEngineering, Nanyang Technological University, Singapore

Glaucoma is a progressive optic neuropathy, which, if left untreated, leads to blindness. The most common and most modifiablerisk factor in glaucoma is elevated intraocular pressure (IOP), which can be managed surgically by filtration surgery. The postoper-ative subconjunctival scarring response, however, remains the major obstacle to achieving long-term surgical success. Antiprolifer-atives such as mitomycin C are commonly used to prevent postoperative scarring. Efficacy of these agents has been tested ex-tensively on monkey and rabbit models of glaucoma filtration surgery. As these models have inherent limitations, we havedeveloped a model of glaucoma filtration surgery in the mouse. We show, for the first time, that the mouse model typically scarredwithin 14 d, but when augmented with mitomycin C, more animals maintained lower intraocular pressures for a longer period oftime concomitant with prolonged bleb survival to beyond 28 d. The morphology of the blebs following mitomycin C treatment alsoresembled well-documented clinical observations, thus confirming the validity and clinical relevance of this model. We demon-strate that the antiscarring response to mitomycin C is likely to be due to its effects on conjunctival fibroblast proliferation, apopto-sis and collagen deposition and the suppression of inflammation. Indeed, we verified some of these properties on mouse conjunc-tival fibroblasts cultured in vitro. These data support the suitability of this mouse model for studying the wound healing response inglaucoma filtration surgery, and as a potentially useful tool for the in vivo evaluation of antifibrotic therapeutics in the eye.© 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.orgOnline address: http://www.molmed.orgdoi: 10.2119/molmed.2010.00188

Address correspondence and reprint requests to Li-Fong Seet, Singapore Eye Research In-

stitute, 11 Third Hospital Avenue, #06-00 SNEC Building, Singapore 168751. Phone: (65)

63275812; Fax: (65) 63224599; E-mail: [email protected]; or Tina T Wong, Glaucoma

Service, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751.

Phone: (65) 63227477; Fax: (65) 62277290; E-mail: [email protected].

Submitted October 1, 2010; Accepted for publication January 6, 2011; Epub

(www.molmed.org) ahead of print January 11, 2011.

although simplified models for the studyof subconjunctival wound healing havebeen reported previously (18,19). Amouse model of GFS would be invalu-able for several reasons. First of all,many critical reagents such as antibodiesare available for the mouse to study thewound healing response by histology,flow cytometry, enzyme-linked im-munosorbent assay (ELISA), and so on,and in vivo ocular imaging techniquesare readily accessible. Understanding thewound healing or fibrotic process is cru-cial to the discovery of novel antifibrotictherapeutics. Secondly, the mapped andsequenced mouse genome makes possi-ble the manipulation of target genes inthe mouse, facilitating the design ofoverexpression DNA constructs, microRNA or small interfering RNAmolecules as potential therapeutics to bevalidated first in the mouse. Thirdly,many potential therapeutics such as neu-tralizing antibodies or antagonizingreagents specifically for the mouse areavailable, allowing proof-of-concept ex-periments to be performed readily.Fourthly, the GFS response in geneticallyaltered animals such as knockouts ortransgenic mice can be easily studied.

We have developed a mouse model ofGFS which can be employed to investi-gate the postoperative subconjunctivalscarring response (21). To ascertain thatthe murine model is relevant to the clini-cal setting, we applied MMC in the mousemodel and examined the postoperativesurgical responses. MMC is commonlyapplied intraoperatively to reduce thepostoperative scarring response follow-ing filtration surgery in patients. We in-vestigated the effect of intraoperativeMMC on bleb survival and morphologyby incorporating major imaging tech-niques used in the clinic, including slitlamp examination, anterior segment- optical coherence tomography (AS-OCT)as well as in vivo confocal imaging. Wealso examined the pathohistology of theblebs with and without MMC treatmentby polarization microscropy and im-munofluorescent analyses. These werefurther corroborated by in vitro studies

using cultured mouse conjunctival fi-broblasts. We provide data to show notonly the mechanisms whereby MMCmay improve surgical success in GFS,but at the same time, the probable rea-sons for the known side effects attributedto MMC in humans. Most importantly,we demonstrate that our mouse model issuitable, valid and closer to the actualhuman glaucoma filtration surgery thanother hitherto described mouse modelsof conjunctival wound healing.

MATERIALS AND METHODS

Mouse Model of Glaucoma FiltrationSurgery

C57BL/6 mice were bred and main-tained at the Singhealth ExperimentalMedical Centre (Singapore General Hos-pital, Singapore). All experiments withanimals were approved by the Institu-tional Animal Care and Use Committee(IACUC) and treated in accordance withthe Association for Research in Visionand Ophthalmology (ARVO) Statementon the Use of Animals in Ophthalmicand Vision Research. The mice wereanesthetized by intraperitoneal (i.p.) in-jection of a 0.1 mL ketamine/xylazinemixture containing 2 mg/mL xylazinehydrochloride (Troy Laboratories, Smith-field, Australia) and 20 mg/mL ketaminehydrochloride (Ketamine, Parnell Labo-ratories, Alexandria, Australia) beforethe operation was carried out. The modi-fied filtering surgery was performedonly on the left eye of each mouse as de-scribed in the text. MMC (Kyowa HakkoKirin Co. Ltd, Shizuoko, Japan) was ap-plied at 0.4 mg/mL with a small piece ofsurgical sponge (MQA, Inami, Tokyo,Japan). Irrigation of the MMC-treatedarea was performed with 2 mL of 0.9%sodium chloride (B Braun MelsungenAG, Melsungen, Germany) using a sy-ringe. The dissected conjunctiva was se-cured and closed by an 11-0 (0.1 metric)Ethilon monofilament nylon scleral su-ture (Ethicon Inc., Somerville, New Jer-sey, USA). Fucithalmic ointment (LeoPharmaceutical Products, Ltd, PrincesRisborough, Buckinghamshire, UK) was

instilled at the end of the procedure.MMC treatment was performed on eighteyes, and control (without treatment)was performed on 10 eyes.

Measurement of IOPThe mice were anesthetized as de-

scribed above before IOP measurements.Intraocular pressures were recorded inboth eyes of each animal with a handheldcommercial rebound tonometer accordingto instructions by the manufacturer(TonoLab, Icare, Espoo, Finland). Allmeasurements were taken between 4 and7 min after anesthetic injection, as sug-gested by a prior study (20); 5 to 10 meas-urements of IOP in each eye were takenand the mean value of the IOP of the leftoperated eye is expressed as a percentageover that of the unoperated right eye ofthe same animal. An IOP that is < 70% ofbaseline IOP is used as an indicator of ef-fective filtration and bleb function. Eightanimals from each group were subjectedto IOP measurements.

Detection and Analysis of BlebsCareful slit lamp, anterior segment-

optical coherence tomography (AS-OCT)and in vivo confocal microscopic exami-nations on subconjunctival blebs wereperformed at postoperative day 2, andweekly thereafter for a total of 4 wks asdescribed previously (21). A bleb wasjudged to have failed if the surgical siteappeared flat by slit lamp analysis. Slitlamp examination was performed bythree examiners who were masked tothe treatment groups. All animals fromeach group were subjected to slit lampand AS-OCT analyses. In vivo confocalmicroscopy was performed on two ani-mals from each group as described previously (21).

Histology and ImmunofluorescentAnalyses

Mice were euthanized on day 28 aftersurgery, and the eyes were enucleated forimmediate fixation and processing as de-scribed (21). Five-μm sections werestained with hematoxylin and eosin to vi-sualize tissue morphology. To assess the

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M I T O M Y C I N C A N D G L A U C O M A F I L T R A T I O N S U R G E R Y

collagen matrix, picrosirius red stainingwas performed as described previously(22) and visualized by polarization mi-croscopy (Olympus BX51, Olympus, Cen-ter Valley, PA, USA). For immunofluores-cent analysis, antibodies specific forcollagen (Abcam plc, Cambridge, UK),Ki67 (Abcam plc), CD45 (BD Pharmingen,Lexington, KY, USA), CD11b (Chemicon,Temecula, CA, USA) and α-SMA (Abcamplc) were used. The primary antibodieswere visualized using secondary antibod-ies conjugated to either AlexaFluor-488 orAlexaFluor-594 (Invitrogen, Eugene, OR,USA). Sections were visualized using theZeiss Imager.Z1 microscope (Carl ZeissMicroImaging GmbH, Göttingen, Ger-many). Histology was performed on threeeyes from each group.

Terminal Deoxynucleotidyl TransferasedUTP Nick End Labeling (TUNEL)Analysis

End labeling of exposed 3′-OH ends ofDNA fragments in cryosections and cul-tured mouse conjunctival fibroblasts wasperformed with the DeadEnd Fluoromet-ric TUNEL System according to manu-facturer’s instructions (Promega, Madi-son, WI, USA). Staining of the cellnucleus was achieved by mounting theTUNEL-stained cryosections or fibrob-lasts in DAPI-containing Vectashieldmounting medium (Vector Laboratories,Burlingame, CA, USA). Sections andcells were visualized using the Zeiss Im-ager.Z1 microscope (Carl Zeiss Inc.).

Cell Culture and Treatment with MMCConjunctival fibroblasts obtained from

C57BL6/J mice were cultured as de-scribed previously (21). For treatmentwith MMC, cells were treated with asingle application of 0.4 mg/mL MMC for1 min. After treatment, cells were washed3× with phosphate-buffered saline (PBS)(Invitrogen) and maintained in culturemedium for 72 h before analyses.

Real-Time Cell Proliferation AnalysisThe xCelligence real-time cell analyzer

(Roche Diagnostics GmbH, Penzberg,Germany) was used to assess cell prolifer-

ation according to manufacturer’s instruc-tions. Mouse conjunctival fibroblasts weretrypsinized and seeded at 8,000 cells perwell in an E-Plate 96 (Roche) in quadru-plicates. For cells treated with MMC, drugtreatment was performed at 0.4 mg/mLfor 1 min on trypsinized cells followed bythree washes in PBS before being seededonto the wells at 8,000 cells/well in nor-mal culture medium. The plated cellswere allowed to equilibrate for at least 30min in the tissue culture incubator beforeelectrode resistance was recorded. Cellgrowth was monitored continuously forup to 6 d.

RNA Isolation and ExpressionTotal RNA recovery, first-strand cDNA

synthesis and quantitative real-time PCR(qPCR) was performed as described pre-viously (21). All PCR reactions were per-formed in triplicate. All mRNA levelswere measured as CT threshold levels andwere normalized with the correspondingβ-actin CT values. Values were expressedas fold increases over the correspondingvalues for untreated control by the 2ΔΔCT

method. The primers for collagen Iα1 andβ-actin were as described previously (21).The presented data is representative ofthree independent experiments.

Statistical AnalysisAll data were expressed as mean ±

standard deviation (SD) where appropri-ate. Survival analysis was performed forbleb failure using the Kaplan-Meier log-rank test. The significance of differencesamong groups was determined by theone-tailed Student t test using the Mi-crosoft Excel 5.0 software. P < 0.05 wasconsidered statistically significant.

RESULTS

A Murine Model of GlaucomaFiltration Surgery

Modified filtering surgery in themouse was undertaken on the left eye asdescribed previously (21) (Figure 1). Thegoal of the surgery was to create a fistulathrough the sclera to facilitate the out-flow of aqueous humor from the anterior

chamber into the subconjunctival space,and thereby cause a reduction in the IOP.

MMC Treatment Was More Effective inLowering IOP

Absolute IOP values do not take intoaccount IOP variability of the individualmouse, as was observed in humans (23).To better assess the impact of the surgeryon IOP, we expressed the IOP of the oper-ated left eye as a percentage of the IOP ofthe unoperated right eye of each animal ateach time point measurements weretaken. Errors arising from variability inindividual baselines are thus eliminatedand the presented data is reflective of theIOP alteration from the baseline that ischaracteristic of each animal as a result ofthe surgery. As shown in Figure 2A, themean % IOP of the operated versus thepaired unoperated eye in the control ani-mals reduced from 99% ± 14% on day 7 to65% ± 20% on day 14, and then rose backup to 84% ± 28% on day 21 and 85% ±14% on day 28. This profile suggests thata reduction in IOP in the operated eyes ismaximal in the second week after surgerywith a return to baseline thereafter. Treat-ment with MMC produced a sustained re-duction in the IOP of the operated mice,with the mean % IOP dropping to 70% ±21% on day 7, and this was maintainedfor the next 3 wks (% IOP = 71% ± 16%,74% ± 19% and 75% ± 26% on days 14, 21and 28 respectively). Since a target pres-sure of 20% to 30% lower than baseline isrecommended for most glaucoma patients(23), we analyzed the efficacy of MMCtreatment in this model in achieving a30% reduction from baseline IOP (equiva-lent to a % IOP of operated versus unop-erated eye of 70%). As shown in Figure2B, MMC treatment resulted in 50% of theoperated mice compared with none in thecontrol group achieving an IOP that was70% of the baseline IOP on day 7. Atday 14, an equal number of the control orMMC-treated animals had a 30% reduc-tion in IOP (50% in each group). In thethird and fourth week after surgery, twicemore MMC-treated animals achieved a30% IOP reduction compared with controlanimals. The number of eyes exhibiting a

R E S E A R C H A R T I C L E


reduction in IOP to less than 70% of theunoperated baseline is maximal on day 14(4 of 8 eyes) in control eyes and day 21 inMMC-treated eyes (6 of 8 eyes) and this isevident as a shift in the profile in Figure2B. MMC treatment is thus more effectivethan control in achieving a higher fre-quency of a reduction in IOP by 30% ormore.

MMC Prolonged Bleb SurvivalFiltration of fluid from the anterior

chamber through the surgically createdfistula into the subconjunctival space isobvious as an elevated conjunctival bleb.The raised, filtering blebs were conspicu-ous in the initial 48 h following surgeryin both the control and MMC-treatedeyes, as analyzed by slit lamp microscopy

(Figure 3A). While the blebs in the MMC-treated eyes remained obvious on day 7after surgery, the blebs in the control eyesbecame less elevated and noticeably morevascularized. By day 14 after surgery,there was no obvious filtering bleb in thecontrol eyes, as evidenced by the flat-tened, vascularized conjunctiva at the sitewhere the bleb was previously noted. Incontrast, the MMC-treated eyes retainedthe presence of a bleb that appeared cys-tic and less vascularized on day 28. In-deed, a significant difference in bleb sur-vival was observed between the twogroups (log rank; P = 0.013; Figure 3B).With MMC, 6 of 8 blebs (75%) survived atday 28. This is in contrast to control eyeswhere only 1 of 10 blebs (10%) survivedat the end of the study. No significant ad-

verse clinical complications or MMC toxi-city such as corneal erosion, cornealopacification, cataract, endophthalmitis,or moderate to severe inflammation on



Figure 1. A murine model of glaucoma filtration surgery. Schematic diagram depicting thesurgical process in the mouse eye. The conjunctiva was dissected so as to expose thesclera and a small filtration subconjunctival space was created by running the surgicalscissors underneath the dissected conjunctiva (A). An incision was then made with a 30-gauge needle through the sclera into the anterior chamber of the eye to create a fistula(B) so as to allow aqueous humor to exit from the anterior chamber and into the subcon-junctival space (C). The final surgical step involved suturing the dissected conjunctivaover the newly created fistula (D). For the MMC treatment arm, 0.4 mg/mL MMC was ap-plied between the sclera and the dissected conjunctiva using a small section of surgicalsponge for 1 min (E). After the sponge was removed, the area was irrigated with 0.9%sodium chloride before cannulation of the sclera (B) and the final suturing of the con-junctiva (D). This figure is modified from a similar figure published previously (21).

Figure 2. Increased number of eyes withintraoperative episcleral application ofMMC maintained a lower IOP than controleyes. (A) The IOPs of both operated andunoperated eyes of each animal at theindicated postsurgery day were measuredat least five times. Each value representsthe % mean IOP of the operated eye overthe mean IOP of the unoperated eye ofthe same animal at each time pointmeasurements were taken. Each symbolrepresents a single animal (, control, n =8; , MMC-treated, n = 8). The horizontalbars and numbers indicate the mean %IOP. Dotted line indicates the 70% IOPlevel. C indicates control. (B) Graphicalrepresentation of the fraction of eyes withIOP maintained at less than 70% of the un-operated IOP of the same animal as afunction of time after surgery. The valueswere calculated on the basis of panel A.

the sclera, were recorded in any of theeyes from each group.

Further analysis by anterior segment-optical coherence tomography (AS-OCT)was performed to evaluate bleb survival.OCT measurements on days 2, 7, 14, and28 after surgery confirmed the slit lamp

observations (Figure 4A). The bleb in arepresentative control operated eye canbe observed to reduce progressively insize until its complete disappearance byday 14. In contrast, the bleb in a repre-sentative MMC-treated eye remained vis-

ible on each of the indicated days for 28 d (see Figure 4A).

In vivo confocal microscopy of the con-trol blebs on day 2 revealed the presenceof optically clear spaces filled with fluid(Figure 4B, ii) which were absent in the



Figure 3. Augmentation of glaucoma filtra-tion surgery with MMC prolonged bleb sur-vival. (A) Slit lamp examination of the sur-gical sites revealed the presence of theblebs (arrowheads) in the conjunctiva ofthe operated eyes. Ten control and eightMMC-treated eyes were examined. (B)Kaplan-Meier survival curve showing thatsurvival rates of filtering bleb were im-proved with MMC treatment. Bleb survivalwas determined on the basis of slit lampexamination as shown in (A). Ten controleyes and eight MMC-treated eyes werescored, P = 0.013 (Kaplan-Meier log-ranktest).

Figure 4. In vivo imaging of the surgical sites. (A) Anterior segment optical coherence to-mography imaging of the mouse conjunctiva. The unoperated and operated eyes on days 2, 7, 14 and 28 were examined. The location of the bleb is indicated by the whitebox. Images shown belong to the same control or MMC-treated eye over the time courseof the analysis. Ten control eyes and eight MMC-treated eyes were examined. C indicatescornea. (B) In vivo confocal microscopy images of the blebs. (i, v) The subconjunctivalmatrix of the unoperated eye is indicated by a white *. Numerous microcysts in the con-junctival matrix can be observed in both control (ii) and MMC-treated (vi) eyes 2 d aftersurgery. While the dull microdots in the control eye are likely to be inflammatory cells de-rived from the surrounding vascular or lymphatic network (iii), MMC treatment is associ-ated with the presence of hyperreflective microdots, which may represent necrotic ep-ithelial cells (vii). (iv) By day 28 in the control eye shown, the operated site appearedscar-like with a dense connective tissue with few or no clear spaces (black *). (viii) TheMMC-treated operated site maintained numerous microcysts although some encapsula-tion may be present at the periphery of the bleb. Two eyes from each group were exam-ined. CE, conjunctival epithelium; S, sclera.

unoperated eye (Figure 4B, i). Thesespaces may correspond with microcystswhich were reported to be numerous infunctioning human blebs (24). The sub-conjunctival connective tissue also ap-peared to be arranged loosely in the con-trol day 2 bleb (Figure 4B, ii, iii). Likewise,we observed numerous well-defined clearspaces in the blebs of the MMC-treatedeyes that are reminiscent of microcysts(Figure 4B, vi, vii). Interestingly, we alsoobserved the presence of hyperreflectivemicrodots in the bleb of the MMC-treatedeye (see Figure 4B, vii). On day 28, thesubconjunctival space in the flattenedbleb area of the control eye was observedas an optically dense, fibrotic connectivetissue with no clear spaces (Figure 4B, iv).In contrast, relatively large microcysticspaces remained in the bleb of the MMC-treated eye and these were surrounded byloosely organized connective tissue (Fig-ure 4B, viii). Optically dense fibrotic tissueadjunct to the conjunctival epitheliumthat is evocative of encapsulation was alsoobserved in the MMC-treated bleb (seeFigure 4B, viii).

MMC Reduced Collagen Depositionin the Bleb

We next investigated the histopatho-logical differences underlying thewound response between control andMMC-treated eyes at day 28 after sur-gery. The control surgical site comprisedof thick strands of fibrous material in thesubconjunctival space overlying thesclera, which consisted of even denserand more tightly packed connective tis-sue (Figure 5A, B). In comparison, thesurgical site in the MMC-treated eye wasvacuous and punctuated with thin, finestrands of connective tissue fibrils (Fig-ure 5C, D). These observations suggestthat collagen deposition might be alteredin the MMC-treated eyes.

To assess the collagen deposition in thecontrol versus MMC treatment, we per-formed sirius red polarization mi-croscopy on the tissue sections. Indeed,the surgical site in the control eye wasdensely compacted with thick, well-aligned collagen fibers resembling a scar

(Figure 5E, F). Moreover, the controlwound site showed a predominance ofmature collagen fibers which were orange-red birefringent (see Figure 5F). Incontrast, the bleb in the MMC-treatedeyes contained few and thin, loosely as-sembled collagen fibers in an expandednoncollagenous subconjunctival space(Figure 5G, H). The majority of the colla-gen fibers also were noticeably yellow-

green birefringent in the MMC-treatedbleb, suggesting the preponderance ofimmature fibers (see Figure 5H). Thus,the survival of the bleb in the MMC-treated eye is, in part, due to reduceddeposition of collagen fibers and deficientmaturation of the scar at the wound site.

To further determine differences inthe extracellular matrix of the controland MMC-treated conjunctiva, immuno-



Figure 5. MMC reduced collagen deposition at the surgical site. (A,C) Hematoxylin andeosin (H&E) staining revealed a flattened conjunctiva at the day-28 control operated site(boxed area) whereas the MMC-treated operated site subconjunctival space was ex-panded with an almost clear matrix (boxed area); scale bar, 500 μm. (B,D) Higher magni-fication of insets (boxed areas in A and C respectively); scale bar, 100 μm. (E,G) Picrosiriusred-stained sections of the same eyes at low magnification and visualized by brightfieldmicroscopy; scale bar, 100 μm. (F,H) Higher magnification of insets (boxed areas in E andG respectively) and polarization microscopy revealed differences in the collagen matri-ces of the control versus MMC-treated eye; scale bar, 100 μm. (I,J) Immunofluorescenceanalysis of sections from the same eyes with a collagen I–specific antibody; scale bar,10 μm. S, sclera.

fluorescence analysis for collagen ex-pression was carried out. The control-operated conjunctiva exhibited more en-hanced staining for collagen I in thesubconjunctival space (Figure 5I) whencompared with the MMC-treated coun-terpart (Figure 5J).

MMC Inhibited Proliferation andInduced Apoptosis in the MouseOperated Conjunctiva

The presence of proliferative cells atthe surgical site was visualized by im-munostaining the cryosections with anti-bodies against Ki67 (25). Surprisingly, atday 28 after surgery, Ki67-positive cellscan be observed close to the edge of thewound in the control conjunctival epithe-lium, suggesting that there are possiblywound-activated proliferating cells re-maining at the surgical site (Figure 6A,upper panel, arrowheads). As expected,there were no visible proliferating cells atthe MMC-treated wound site (Figure 6A,bottom panel).

To determine the induction of apopto-sis by MMC, cryosections were subjectedto TUNEL staining. The control conjunc-tiva did not present with any TUNEL-positive cells (Figure 6B, upper panel). Instriking contrast, almost all of the cells inthe conjunctival epithelium as well ascells in the subconjunctival space andthose abutting the episclera were apop-totic in the MMC-treated surgical site(Figure 6B, lower panel, arrowheads).This finding raises concern about tissuerecovery, especially of the conjunctivalepithelium, since the presence of apop-totic cells here seemed to persist for atleast 28 d after surgery and there wereno apparent proliferative cells to repaireventual cell loss due to apoptosis.

MMC Suppressed the Recruitment ofInflammatory Cells to the Surgical Site

The influx of inflammatory cells to thesurgical site is a wound healing responseassociated with fibrosis. To examine this,tissue sections were subjected to im-munostaining for CD45 and CD11b ex-pressions. CD45, present on the surfaceof nucleated cells including B cells,

T cells, neutrophils and macrophages ofhematopoietic origin, is commonly usedas a marker for inflammation (26). Theconjunctival epithelium, subconjunctivalspace as well as the most anterior celllayer of the sclera at the wound site ofthe control eye revealed positive stainingfor CD45 (Figure 7A, arrowheads). Incontrast, CD45 expression was detectedmainly in the more posterior cell layersof the sclera in the bleb of the MMC-treated eye (Figure 7B, arrowheads).CD11b, a cell surface marker expressed athigh levels on macrophages and mono-cytes (27), was found to be expressed inthe episclera of the control wound site(Figure 7C, arrowheads) but absent inthe MMC-treated eye (Figure 7D). These

observations may be explained by thepossibility that MMC caused the loss ofnormal conjunctival and episcleral vas-culatures so that recruitment of CD45+

and CD11b+ cells to the conjunctival ep-ithelium and episclera was suppressed.To examine this possibility, we carriedout immunofluorescent analysis of con-secutive tissue sections with antibodiesagainst α-SMA. First of all, we did notobserve any fibroblasts in the conjunctivaexpressing this marker in either controlor MMC-treated eyes. This is possiblydue to the fact that by day 28, the woundhealing process is at a late stage, and α-SMA-positive myofibroblasts areknown to be lost after the early activeperiod of wound contraction (28,29). In-



Figure 6. Cell death in the conjunctiva and episclera after MMC treatment. (A) Tissue sec-tions were stained with Ki-67 (red) to visualize proliferating conjunctival epithelial cells inthe control operated eye (arrowheads) versus the absence of any detectable proliferat-ing cells in the MMC-treated operated eye; scale bar, 100 μm. (B) No apoptotic (TUNEL-positive) cells, which would have appeared as bright green cells, were seen in the controloperated site, while many apoptotic cells were observed in the conjunctival epitheliumas well as the episclera at the MMC-treated surgical site (arrowheads); scale bar, 10 μm.

stead, we observed α-SMA-positive cellspresent around structures that resembledblood vessels (Figure 7E, F, arrowheads).α-SMA is known to be expressed byperivascular mural cells or pericytes (30).Interestingly, the locations of these po-tential blood vessels were close to theepisclera in the control surgical site (Fig-ure 7E). This would account for the pres-ence of inflammatory cells near the epis-clera of the surgical site in the controleye. In contrast, we observed few α-SMA-positive blood vessel-like struc-tures in the MMC-treated sclera and,when they could be observed, they ap-

peared to exist in the deeper more poste-rior scleral layers (Figure 7F, arrowhead).This may explain why CD45+ cells wereobserved mainly in the more posteriorscleral layers and not in the episclera ofthe MMC-treated site.

MMC Inhibited Proliferation, InducedApoptosis and Inhibited Collagen Iα1Expression in Cultured MouseConjunctival Fibroblasts

To verify the mechanism of action ofMMC deduced from the in vivo observa-tions described above, as well as to de-termine if the observed in vivo effects

parallel the in vitro cellular response toMMC treatment, we performed a seriesof experiments on cultured mouse con-junctival cells. Using the real time cellanalyzer (RTCA) SP instrument (31), weanalyzed conjunctival cell growth afterMMC treatment in comparison to controluntreated cells. Cell index values pro-duced by the MMC-treated cells indi-cated the lack of proliferation, while thecell index values of the control cells increased progressively with time (Fig-ure 8A). Hence, a single application ofMMC was able to suppress the growth ofthe mouse conjunctival fibroblasts effec-tively for up to 6 d. The lack of prolifera-tion was verified by the lack of Ki67 pos-itive staining in cells that were treatedwith MMC (Figure 8B, lower panel) com-pared with control cells (Figure 8B,upper panel). When analyzed by TUNELstaining, only a small number of MMC-treated cells were observed to be apop-totic (Figure 8C, lower panel, arrow-heads) while none were observed in thecontrol (Figure 8C, upper panel). Thiswas surprising considering that most ofthe cells in the MMC-treated conjunctivain vivo appeared TUNEL positive. TheMMC-treated cells also were examinedfor the expression of collagen Iα1 mRNAby qPCR. MMC treatment significantlyand consistently reduced collagen Iα1mRNA expression compared with con-trol (Figure 8D).

DISCUSSIONWe confirm in this study the validity

of a model for GFS in the mouse. To ourknowledge, the closest known mousemodel for GFS is a simple conjunctivalscarring model where a fixed volume ofsaline was injected into the subconjuncti-val space to create a visible bleb (19).Our model is significantly closer to thesurgical procedure, known as a tra-beculectomy that is performed on humaneyes. The major surgical endpoint of atrabeculectomy is a filtering bleb allow-ing aqueous humour to flow from theanterior chamber into the subconjuncti-val space. This endpoint is achieved withour mouse model. The only major differ-



Figure 7. Recruitment of inflammatory cells to the operated site of control versus MMC-treated eyes. (A,B) Immunostaining for CD45+ leukocytes revealed the presence of thesecells in the conjunctival epithelium and episclera region of the control operated area (A, arrowheads) while in the MMC-treated operated site, they were detected mainly inthe posterior scleral cell layers (B, arrowheads). (C,D) Immunostaining with the CD11b an-tibody showed that inflammatory cells were present in the episclera area of the controloperated site (C, arrowheads) while CD11b-positive inflammatory cells were absent inthe MMC-treated operated area. (E,F) α-SMA-positive cells present in the episcleralarea of the control operated site may represent pericytes surrounding vascular vessels(E, arrowheads) which in the MMC-treated eye were present only in the posterior scleralcell layers at the surgical site (F, arrowhead). CE, conjunctival epithelium; S, sclera. Scalebar, 10 μm.

ence in the mouse surgical model is that,unlike in trabeculectomy, a partial thick-ness guarded scleral flap is not createdowing to the very thin sclera of themouse eye, causing this step of the oper-ation to be technically and surgicallyvery challenging. Therefore needle tractsclerostomy, which scarred in 14 days,was performed instead, as described inthis study. The intraoperative applicationof MMC extended the bleb survival pe-riod to beyond 28 days, supporting theuse of this drug in the clinic to improvesurgical success. We also demonstratedthat treatment with MMC resulted in ahigher frequency of surgical successbased on maintenance of an IOP below70% of baseline. These two MMC- induced effects in our mouse model par-allel those observed previously in therabbit (32) and the monkey (33) models.Furthermore, alterations in the in vivobleb structure in our model were remi-niscent of those observed previously inthe clinic (24). Histologic features alsomirrored those observed in the clinicwith reduced matrix deposition in theMMC eyes compared with the controleyes. Given the close correlation withdocumented clinical response, our modelis ideal not only for studying the healingand fibrotic processes that are activatedfollowing surgery, but also for delineat-ing the mode of drug action.

In the present study, we evaluated theeffect of MMC on three aspects of con-junctival wound healing in the mouseGFS model: cell proliferation, depositionof collagen and inflammation in thewound bed. As in trabeculectomies (34),the majority of the scarring effects in ourmouse model were observed at the levelof the episclera and the subconjunctivalspace.

The profound cytotoxic effect of MMCin the proliferation phases of Tenon’s fi-broblasts (35–37) was demonstratedclearly in our model where the MMC-treated conjunctiva contained no detect-able proliferative cells compared withthe control wound site, which did con-tain proliferative cells. Mouse conjuncti-val fibroblasts cultured in vitro showed



Figure 8. MMC effects on mouse conjunctival cell proliferation, apoptosis and collagenIα1 expression. (A) Cell growth was monitored continuously using the RTCA SP instrument.The cell index profiles of untreated cells (dotted black line) and MMC-treated cells (redline) reflect the logarithmic growth phase and response to treatment respectively. The cellindex values for MMC-treated cells did not demonstrate a logarithmic growth phase thatwas measured for the control cells indicating that the treated cells did not proliferate. (B)Cells were treated with MMC for 1 min and maintained in culture medium for 72 h beforestaining with DAPI (blue) and anti-Ki67 antibody (red). Selected cells that are positive forKi67 are indicated by arrowheads; scale bar, 100 μm. (C) Cells were treated as in (B) andanalyzed by TUNEL staining (green). Nuclei were visualized by DAPI staining (blue). Cellspositive for TUNEL staining are indicated by arrowheads; scale bar, 100 μm. (D) Cells weretreated the same as in (B) and assayed for collagen Iα1 mRNA abundance by real-timequantitative PCR. MMC treatment significantly reduced the amount of collagen Iα1mRNA by 40% compared with untreated control cells (P = 1.8 × 10–10). Bars represent theSD. Data shown is representative of three independent experiments.

the same antiproliferative response toMMC. It has been suggested that MMCprevents the recruitment and activationof conjunctival fibroblasts through the in-duction of apoptosis in these cells (38).Indeed, the majority of the cells in theMMC-treated conjunctival epithelium,subconjunctival space, as well as thesclera at the wound site, were apoptotic,which was not the case with the controlwound site. The apoptotic effect of MMCalso was observed in cultured mouseconjunctival fibroblasts. However, not allthe cells cultured in vitro were apoptotic,as was also observed in an earlier studyon human Tenon fibroblasts (38). Thus,the question arises as to whether acti-vated conjunctival fibroblasts, which cul-tured cells essentially represent, beingdevoid of inhibitory compact surround-ings, are less sensitive to MMC. This isclinically important because, if MMCcauses indiscriminate apoptosis in bothactivated and inactivated cells, the loss ofconjunctival epithelial and fibroblastcells may cause detrimental conjunctivalthinning after a single application. Ourobservation confirms a previous reportthat MMC effects on rabbit subconjuncti-val and scleral fibroblast growth wasmaintained 30 days after GFS (39). Thismay explain why the use of MMC clini-cally increases the likelihood that blebswill become thin and cystic, which inturn leads to a lifelong risk of conjuncti-val breakdown, aqueous humour leakageand bleb-related infections and endoph-thalmitis (40). Hence, although it hasbeen reported that MMC disappears rap-idly from the ocular tissue and that con-centration of the agent is reduced signifi-cantly by irrigating the tissue copiouslyimmediately after its application (37), themouse model suggests that long-term ef-fects of MMC in vivo should be investi-gated in detail.

MMC is also thought to inhibit colla-gen deposition and disorganization, amajor phenomenon in fibrosis, by the in-duction of apoptosis of conjunctival fi-broblasts (38). While this may definitelyplay a part in vivo, we showed in thisstudy that MMC treatment may also sup-

press collagen I expression independentof apoptosis in vitro. We believe thatapoptotic cells, which were fairly low innumbers in vitro based on TUNEL stain-ing, could not have been the cause forthe significant reduction in collagen ImRNA expression in the presence ofMMC. We speculate that MMC may af-fect collagen I expression via a distinctpathway. Further work is required to ad-dress this issue since regulation of colla-gen production and organization is keyto the inhibition of fibrosis in the con-junctiva (21).

The avascular MMC-treated blebs ob-served in our model suggest that the in-flammatory response, which is linked tofibrosis, may be dampened in the MMC-treated eyes. Indeed, we observed fewerpersisting inflammatory cells at the oper-ated site after MMC treatment comparedwith control eyes. We believe this is duein part to the suppression of angiogene-sis at the wound site by MMC, which isknown to have potent antiangiogenicproperties (39). While a microvascularnetwork was apparent around the oper-ated site soon after wounding in the con-trol mouse eye, the MMC-treated blebswere characterized by the lack of vascu-lature which corresponded with similarobservations in the clinic (41). The sup-pression of angiogenesis is, however, adouble-edged sword. On the one hand,inhibiting angiogenesis may limit tissuescarring, but on the other, the lack of aprotective inflammatory response stem-ming from lack of vasculature may con-tribute to an increased risk of infection atthe operated site.

In conclusion, this study has demon-strated that MMC can delay wound heal-ing or fibrosis by mechanisms beyondthe inhibition of proliferation. However,the sustained apoptotic effect of MMC onthe conjunctival epithelium, subconjunc-tiva and episclera should be cause forconcern particularly with respect to con-junctival breakdown and risk of infectionin the long term when used in conjunc-tion with GFS. Finally, the comparablesimilarities between the clinical effects ofMMC and that observed in our mouse

model not only highlighted the suitabil-ity of this model for studying the surgi-cal response to known therapeutics butalso indicated the application of thismodel as an invaluable platform for anin-depth understanding of the woundhealing response per se which will in turnfacilitate the discovery and testing ofnovel antifibrotics.

ACKNOWLEDGMENTSWe thank Hla Myint Htoon (Singapore

Eye Research Institute) for help with thestatistical analysis and the Department ofPathology, Yong Loo Lin School of Medi-cine, National University of Singapore,for help with the polarizing microscopy.This work was supported by the Na-tional Research Foundation CouncilTranslational and Clinical Research(TCR) Programme Grant (NMRC/TCR/002-SERI/2008) and a research grantfrom the National Medical ResearchCouncil (NMRC/EDG/0019/2008) to TT Wong.

DISCLOSURESThe authors declare that they have no

competing interests as defined by Molecu-lar Medicine, or other interests that mightbe perceived to influence the results anddiscussion reported in this paper.

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Validation of the Glaucoma Filtration Surgical Mouse Model